Evidence suggests that matrix metalloproteinase (MMPs) play important, albeit frequently paradoxical, roles in multiple pathologies, including cancer, neuropathic pain, chronic wounds, hypertension, and inflammatory diseases. It is an urgent need to develop selective and efficient inhibitors of individual MMPs for biomedical research and disease therapies. However, the catalytic domains of MMPs share a high degree of sequence and fold homology, and thus distinguishing among MMPs using small molecule inhibitors is exceedingly difficult. Because of their exquisite specificity, antibody-based inhibitrs are emerging as promising MMP- blocking agents. Unfortunately, to date, at least two major obstacles make the routine discovery of MMP- inhibiting mAbs difficult: (1) low antigenicity of the MMP active site, and (2) lack of function-based selection methods. Our long-term goal is to develop therapeutic mAbs that would inhibit specific MMPs in disease. The objectives of this project are to (1) overcome these obstacles and establish general, streamlined methodologies for the discovery and engineering of inhibitory antibodies; and (2) use these MMP inhibitory mAbs to advance our understandings on mechanisms of cancer cell migration and test their therapeutic potentials in xenograft models. Our central hypotheses are (1) convex antigen-binding sites (paratopes) are inhibitory; (2) development of a quantitative, function-based high-throughput screening (HTS) greatly accelerates the discovery of inhibitory mAbs; and (3) blocking the specific MMP activities by highly selective functional mAbs inhibits cancer cell migration in vitro and invasion in vivo. Building on our team's expertise in protein engineering, biophysics, cell biology and cancer biology, we will, Aim 1: design, synthesis and optimize human antibody libraries carrying convex paratopes; Aim 2: isolate inhibitory antibodies by function-based high-throughput screening; Aim 3: characterize inhibitory mAbs and elucidate inhibition mechanisms; and Aim 4: test the ability of inhibitory mAbs to alter cancer cell migration in vitro and invasion in vivo. The approaches are innovative, because it will (1) create synthetic human antibody libraries carrying inhibitory paratopes; (2) develop a groundbreaking function-based (rather than binding-based) HTS method for facile discovery of mAbs inhibiting the individual MMPs; (3) uncover how the structure and mechanics of the extracellular matrix controls MMP activity during cancer cell migration and invasion; (4) transform novel antibody-based selective MMP inhibitors into drug leads applicable for biopharmaceutical developments. The proposed research is significant because it will (1) establish a pipeline technology can be readily applied for many biomedically important proteases, one of the largest families of pharmaceutical targets; (2) advance our understanding of the molecular mechanisms by which cancer cells migrate through the extracellular matrix; and (3) initialize the development toward novel immunotherapeutic agents blocking cancer metastasis. Title: Structure-based design of camel-like human selective mAbs against MMPs in disease Keywords: matrix metalloproteinase; inhibitory antibody; synthetic antibody library; camelid antibody; high-throughput screening; complementarity determining region.